The goal of our studies is the elucidation of RNA polymerase II structure, mechanism, and regulation. In previous work, methods were developed for 2-D crystallography of large multiprotein assemblies and applied to RNA polymerase II and its complexes with general transcription factors B and E. The results led to a hypothesis for the transcription initiation mechanism. We now propose the culmination of our efforts, with the structure determination of a complete 28-protein transcription initiation complex, and of an extended initiation complex including 16 additional proteins required for regulation. Structure determination of an actively transcribing complex will reveal the locations of template DNA and product RNA. Specific aims for the project period include the following: 1. Structure determination of RNA polymerase II in ice. 3-D reconstruction from electron micrographs of 2-D crystals is in progress. Extension of resolution by the use of single particle techniques and by combination of phase information from electron micrographs with intensities from X-ray diffraction will be undertaken. 2. Structure determination of the RNA polymerase II initiation complex. A series of RNA polymerase II-general transcription factor complexes will be solved by 2-D crystallography. The results will be combined to yield a picture of a complete RNA polymerase II initiation complex. 3. Structure determination of an RNA polymerase II transcription elongation complex 2-D crystals have been obtained of an actively transcribing complex, containing the polymerase paused at a single base on template DNA, with associated nascent RNA. 3-D reconstruction in ice is in progress. 4. Structure determination of general transcription factor H. a nine-subunit, 500,000 Da complex that possesses two ATPase/helicases and a cdk/cyclin pair, and which shares seven subunits with an even larger DNA repairosome. 5. Structure determination of a regulated RNA polymerase II initiation complex. We will undertake 2-D crystallography of the fifteen- subunit mediator, mediator-polymerase complex, and an extended polymerase II initiation complex, containing promoter DNA with an enhancer sequence, activator protein, mediator, polymerase, and general transcription factors.